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A novel approach using DNA-repair-deficient chicken DT40 cell lines for screening and characterizing the genotoxicity of environmental contaminants.

Ji K, Kogame T, Choi K, Wang X, Lee J, Taniguchi Y, Takeda S - Environ. Health Perspect. (2009)

Bottom Line: We aimed to establish a sensitive and fast screening method that is also capable of characterizing mechanisms of genotoxicity.We also characterized DNA lesions induced by NaAsO(2).Genotoxicity of given stressors was successfully screened based on a comparison of proliferation kinetics between wild-type and DNA-repair-deficient mutants in 48 hr.

View Article: PubMed Central - PubMed

Affiliation: School of Public Health, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Many bacterial or mammalian cell-based test systems, such as the Ames test, chromosomal aberration assays, or gene mutation assays, are commonly used in developed countries to detect the genotoxicity of industrial chemicals. However, the specificity is generally limited and the sensitivity is not sufficiently high. In addition, most assays cannot provide information on mechanisms of genotoxicity of a given chemical.

Objectives: We aimed to establish a sensitive and fast screening method that is also capable of characterizing mechanisms of genotoxicity.

Methods: We developed a novel bioassay employing gene-disrupted clones of the chicken DT40 B-lymphocyte line, which are designed to be deficient in several specific DNA repair pathways. Genotoxic chemicals can delay cellular proliferation in DNA-repair-deficient clones more significantly than in wild-type cells by interfering with DNA replication, thereby inducing DNA damage. In addition, we verified the validity of this assay by analyzing the genotoxicity of gamma-rays, ultraviolet (UV) light, and sodium metaarsenite (NaAsO(2)). We also characterized DNA lesions induced by NaAsO(2).

Results: Genotoxicity of given stressors was successfully screened based on a comparison of proliferation kinetics between wild-type and DNA-repair-deficient mutants in 48 hr. We also found that NaAsO(2) apparently induces at least two types of damage: chromosomal breaks and UV photoproduct-like DNA lesions.

Conclusion: This bioassay is a reliable and sensitive screening tool for environmental mutagens as well as for further characterizing the nature of detected genotoxicity.

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Related in: MedlinePlus

Analysis of cell number by measuring ATP in cellular lysates. (A) The relationship in the range from 102/mL to 106/mL DT40 cells with luciferase activity (the amount of ATP). (B, C) Luciferase activity 24, 48, and 72 hr after irradiation with γ-rays (B) and UV (C); the pattern of cellular proliferation was very similar between 48 and 72 hr after γ-irradiation (B). (D) The cell cycle 6 hr after exposure to 5 J/m2 UV (right) and with no UV irradiation (left) by BrdU pulse-labeling. (E) Amount of luciferase activity per cell. UV irradiation augmented the amount of ATP by 70% at 24 hr after UV compared with nonirradiated cells; 104 cells were exposed to 5 J/m2 UV and subsequently incubated in 5 mL medium for 24 hr.
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f2-ehp-117-1737: Analysis of cell number by measuring ATP in cellular lysates. (A) The relationship in the range from 102/mL to 106/mL DT40 cells with luciferase activity (the amount of ATP). (B, C) Luciferase activity 24, 48, and 72 hr after irradiation with γ-rays (B) and UV (C); the pattern of cellular proliferation was very similar between 48 and 72 hr after γ-irradiation (B). (D) The cell cycle 6 hr after exposure to 5 J/m2 UV (right) and with no UV irradiation (left) by BrdU pulse-labeling. (E) Amount of luciferase activity per cell. UV irradiation augmented the amount of ATP by 70% at 24 hr after UV compared with nonirradiated cells; 104 cells were exposed to 5 J/m2 UV and subsequently incubated in 5 mL medium for 24 hr.

Mentions: To evaluate the cellular response to DNA damage, we exposed wild-type and mutant cells (1 × 103) to various doses of γ-rays and UV light and subsequently incubated them in 1 mL medium. Based on flow cytometric analysis, the number of live cells in each culture was determined 24, 48, and 72 hr after irradiation (Figure 2B,C). We then compared the results of this new protocol with those of a CFA, which is the most reliable standard assay for analyzing cellular response to DNA damage. We also indirectly assessed the number of living cells by measuring the level of ATP in cellular lysates, because this method is deemed more appropriate for high-throughput screening compared with flow cytometric analysis.


A novel approach using DNA-repair-deficient chicken DT40 cell lines for screening and characterizing the genotoxicity of environmental contaminants.

Ji K, Kogame T, Choi K, Wang X, Lee J, Taniguchi Y, Takeda S - Environ. Health Perspect. (2009)

Analysis of cell number by measuring ATP in cellular lysates. (A) The relationship in the range from 102/mL to 106/mL DT40 cells with luciferase activity (the amount of ATP). (B, C) Luciferase activity 24, 48, and 72 hr after irradiation with γ-rays (B) and UV (C); the pattern of cellular proliferation was very similar between 48 and 72 hr after γ-irradiation (B). (D) The cell cycle 6 hr after exposure to 5 J/m2 UV (right) and with no UV irradiation (left) by BrdU pulse-labeling. (E) Amount of luciferase activity per cell. UV irradiation augmented the amount of ATP by 70% at 24 hr after UV compared with nonirradiated cells; 104 cells were exposed to 5 J/m2 UV and subsequently incubated in 5 mL medium for 24 hr.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2801191&req=5

f2-ehp-117-1737: Analysis of cell number by measuring ATP in cellular lysates. (A) The relationship in the range from 102/mL to 106/mL DT40 cells with luciferase activity (the amount of ATP). (B, C) Luciferase activity 24, 48, and 72 hr after irradiation with γ-rays (B) and UV (C); the pattern of cellular proliferation was very similar between 48 and 72 hr after γ-irradiation (B). (D) The cell cycle 6 hr after exposure to 5 J/m2 UV (right) and with no UV irradiation (left) by BrdU pulse-labeling. (E) Amount of luciferase activity per cell. UV irradiation augmented the amount of ATP by 70% at 24 hr after UV compared with nonirradiated cells; 104 cells were exposed to 5 J/m2 UV and subsequently incubated in 5 mL medium for 24 hr.
Mentions: To evaluate the cellular response to DNA damage, we exposed wild-type and mutant cells (1 × 103) to various doses of γ-rays and UV light and subsequently incubated them in 1 mL medium. Based on flow cytometric analysis, the number of live cells in each culture was determined 24, 48, and 72 hr after irradiation (Figure 2B,C). We then compared the results of this new protocol with those of a CFA, which is the most reliable standard assay for analyzing cellular response to DNA damage. We also indirectly assessed the number of living cells by measuring the level of ATP in cellular lysates, because this method is deemed more appropriate for high-throughput screening compared with flow cytometric analysis.

Bottom Line: We aimed to establish a sensitive and fast screening method that is also capable of characterizing mechanisms of genotoxicity.We also characterized DNA lesions induced by NaAsO(2).Genotoxicity of given stressors was successfully screened based on a comparison of proliferation kinetics between wild-type and DNA-repair-deficient mutants in 48 hr.

View Article: PubMed Central - PubMed

Affiliation: School of Public Health, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Many bacterial or mammalian cell-based test systems, such as the Ames test, chromosomal aberration assays, or gene mutation assays, are commonly used in developed countries to detect the genotoxicity of industrial chemicals. However, the specificity is generally limited and the sensitivity is not sufficiently high. In addition, most assays cannot provide information on mechanisms of genotoxicity of a given chemical.

Objectives: We aimed to establish a sensitive and fast screening method that is also capable of characterizing mechanisms of genotoxicity.

Methods: We developed a novel bioassay employing gene-disrupted clones of the chicken DT40 B-lymphocyte line, which are designed to be deficient in several specific DNA repair pathways. Genotoxic chemicals can delay cellular proliferation in DNA-repair-deficient clones more significantly than in wild-type cells by interfering with DNA replication, thereby inducing DNA damage. In addition, we verified the validity of this assay by analyzing the genotoxicity of gamma-rays, ultraviolet (UV) light, and sodium metaarsenite (NaAsO(2)). We also characterized DNA lesions induced by NaAsO(2).

Results: Genotoxicity of given stressors was successfully screened based on a comparison of proliferation kinetics between wild-type and DNA-repair-deficient mutants in 48 hr. We also found that NaAsO(2) apparently induces at least two types of damage: chromosomal breaks and UV photoproduct-like DNA lesions.

Conclusion: This bioassay is a reliable and sensitive screening tool for environmental mutagens as well as for further characterizing the nature of detected genotoxicity.

Show MeSH
Related in: MedlinePlus